Artificial metalloenzymes publications - Ward Group - Department of Chemistry

Host protein:

α3D peptide

Anchoring strategy:

Hydration of C=C and C=O double bonds

Optimization:

Chemical & genetic

Reaction:

Max TON:

---

ee:

---

PDB:

---

Notes:

kcat/KM ≈ 3.8*104 M-1*s-1

A Designed Functional Metalloenzyme that Reduces O2 to H2O with Over One Thousand Turnovers

Lu, Y.

Angew. Chem., Int. Ed., 2012, 10.1002/anie.201201981

Rational design of functional enzymes with a high number of turnovers is a challenge, especially those with a complex active site, such as respiratory oxidases. Introducing two His and one Tyr residues into myoglobin resulted in enzymes that reduce O2 to H2O with more than 1000 turnovers (red line, see scheme) and minimal release of reactive oxygen species. The positioning of the Tyr residue is critical for activity.

Metal:

Ligand type:

Host protein:

Myoglobin (Mb)

Anchoring strategy:

[4Fe-4S] cofactor; Porphyrin

Optimization:

Chemical & genetic

Reaction:

CCO oxidase activity

Max TON:

1056

ee:

---

PDB:

4FWX

Notes:

Sperm whale myoglobin

A Designed Heme-[4Fe-4S] Metalloenzyme Catalyzes Sulfite Reduction like the Native Enzyme

Lu, Y.

Science, 2018, 10.1126/science.aat8474

Multielectron redox reactions often require multicofactor metalloenzymes to facilitate coupled electron and proton movement, but it is challenging to design artificial enzymes to catalyze these important reactions, owing to their structural and functional complexity. We report a designed heteronuclear heme-[4Fe-4S] cofactor in cytochrome c peroxidase as a structural and functional model of the enzyme sulfite reductase. The initial model exhibits spectroscopic and ligand-binding properties of the native enzyme, and sulfite reduction activity was improved—through rational tuning of the secondary sphere interactions around the [4Fe-4S] and the substrate-binding sites—to be close to that of the native enzyme. By offering insight into the requirements for a demanding six-electron, seven-proton reaction that has so far eluded synthetic catalysts, this study provides strategies for designing highly functional multicofactor artificial enzymes.

Metal:

Ligand type:

Host protein:

Cytochrome c peroxidase

Anchoring strategy:

Optimization:

Chemical & genetic

Reaction:

Sulfite reduction

Max TON:

---

ee:

---

PDB:

---

Notes:

Designed heteronuclear heme-[4Fe-4S] cofactor in cytochrome c peroxidase

A Designed Metalloenzyme Achieving the Catalytic Rate of a Native Enzyme

Lu, Y.; Wang, J.

J. Am. Chem. Soc., 2015, 10.1021/jacs.5b07119

Terminal oxidases catalyze four-electron reduction of oxygen to water, and the energy harvested is utilized to drive the synthesis of adenosine triphosphate. While much effort has been made to design a catalyst mimicking the function of terminal oxidases, most biomimetic catalysts have much lower activity than native oxidases. Herein we report a designed oxidase in myoglobin with an O2 reduction rate (52 s–1) comparable to that of a native cytochrome (cyt) cbb3 oxidase (50 s–1) under identical conditions. We achieved this goal by engineering more favorable electrostatic interactions between a functional oxidase model designed in sperm whale myoglobin and its native redox partner, cyt b5, resulting in a 400-fold electron transfer (ET) rate enhancement. Achieving high activity equivalent to that of native enzymes in a designed metalloenzyme offers deeper insight into the roles of tunable processes such as ET in oxidase activity and enzymatic function and may extend into applications such as more efficient oxygen reduction reaction catalysts for biofuel cells.

Metal:

Ligand type:

Host protein:

Myoglobin (Mb)

Anchoring strategy:

Optimization:

Reaction:

O2 reduction

Max TON:

---

ee:

---

PDB:

---

Notes:

O2 reduction rates of 52 s-1 were achieved in combination with the native redox partner cyt b5.

A Designed Supramolecular Protein Assembly with In Vivo Enzymatic Activity

Tezcan, F. A.

Science, 2014, 10.1126/science.1259680

The generation of new enzymatic activities has mainly relied on repurposing the interiors of preexisting protein folds because of the challenge in designing functional, three-dimensional protein structures from first principles. Here we report an artificial metallo-β-lactamase, constructed via the self-assembly of a structurally and functionally unrelated, monomeric redox protein into a tetrameric assembly that possesses catalytic zinc sites in its interfaces. The designed metallo-β-lactamase is functional in the Escherichia coli periplasm and enables the bacteria to survive treatment with ampicillin. In vivo screening of libraries has yielded a variant that displays a catalytic proficiency [(kcat/Km)/kuncat] for ampicillin hydrolysis of 2.3 × 106 and features the emergence of a highly mobile loop near the active site, a key component of natural β-lactamases to enable substrate interactions.

Metal:

Ligand type:

Host protein:

Cytochrome cb562

Anchoring strategy:

Optimization:

Reaction:

Hydrolytic cleavage

Max TON:

---

ee:

---

PDB:

4U9E

Notes:

---

A Hydrogenase Model System Based on the Sequence of Cytochrome c: Photochemical Hydrogen Evolution in Aqueous Media

Hayashi, T

Chem. Commun., 2011, 10.1039/c1cc11157d

The diiron carbonyl cluster is held by a native CXXC motif, which includes Cys14 and Cys17, in the cytochrome c sequence. It is found that the diiron carbonyl complex works well as a catalyst for H2 evolution. It has a TON of ∼80 over 2 h at pH 4.7 in the presence of a Ru-photosensitizer and ascorbate as a sacrificial reagent in aqueous media.

Metal:

Ligand type:

Carbonyl

Host protein:

Cytochrome c

Anchoring strategy:

Optimization:

---

Reaction:

H2 evolution

Max TON:

ee:

---

PDB:

---

Notes:

Horse heart cytochrome C

Alteration of the Oxygen-Dependent Reactivity of De Novo Due Ferri Proteins

DeGrado, W. F.

Nat. Chem., 2012, 10.1038/NCHEM.1454

De novo proteins provide a unique opportunity to investigate the structure–function relationships of metalloproteins in a minimal, well-defined and controlled scaffold. Here, we describe the rational programming of function in a de novo designed di-iron carboxylate protein from the Due Ferri family. Originally created to catalyse the O2-dependent, two-electron oxidation of hydroquinones, the protein was reprogrammed to catalyse the selective N-hydroxylation of arylamines by remodelling the substrate access cavity and introducing a critical third His ligand to the metal-binding cavity. Additional second- and third-shell modifications were required to stabilize the His ligand in the core of the protein. These structural changes resulted in at least a 106-fold increase in the relative rate between the arylamine N-hydroxylation and hydroquinone oxidation reactions. This result highlights the potential for using de novo proteins as scaffolds for future investigations of the geometric and electronic factors that influence the catalytic tuning of di-iron active sites.

Metal:

Ligand type:

Host protein:

Due Ferro 1

Anchoring strategy:

Optimization:

DeGrado, W. F.; Lombardi, A.

Reaction:

N-Hydroxylation

Max TON:

---

ee:

---

PDB:

2LFD

Notes:

---

An Artificial Di-Iron Oxo-Orotein with Phenol Oxidase Activity

Nat. Chem. Biol., 2009, 10.1038/nchembio.257

Here we report the de novo design and NMR structure of a four-helical bundle di-iron protein with phenol oxidase activity. The introduction of the cofactor-binding and phenol-binding sites required the incorporation of residues that were detrimental to the free energy of folding of the protein. Sufficient stability was, however, obtained by optimizing the sequence of a loop distant from the active site.

Metal:

Ligand type:

Host protein:

Due Ferro 1

Anchoring strategy:

Optimization:

Reaction:

Alcohol oxidation

Max TON:

>50

ee:

---

PDB:

2KIK

Notes:

kcat/KM ≈ 1380 M-1*min-1

Metal:

Ligand type:

Host protein:

Due Ferro 1

Anchoring strategy:

Optimization: